Proteomics of early zebrafish embryos

Abstract

BackgroundZebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed.ResultsAs a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins.ConclusionThe provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.

Autotaxin Regulates Vascular Development via Multiple Lysophosphatidic Acid (LPA) Receptors in Zebrafish

“…Western Blotting-Embryos were deyolked as described previously (22) with slight modifications. Briefly, 30 embryos at 48 hpf were dechorionized with forceps and transferred to a 1.5-ml tube.…”

Intraflagellar Transport Proteins Are Essential for Cilia Formation and for Planar Cell Polarity

“…Embryos were manually deyolked following a previously published protocol 41 and then ground in 2ϫ SDS sample buffer (4% SDS, 200 mM dithiothreitol, and 5% ␤-mercaptoethanol) with a disposable pestle, boiled for 5 minutes, cleared by microcentrifugation at top speed at room temperature for 2 minutes, and run on SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes.…”

Lzts2 Regulates Embryonic Cell Movements and Dorsoventral Patterning through Interaction with and Export of Nuclear β-Catenin in Zebrafish

“…Blotting-Embryonic cells were isolated following a previous protocol (52). Co-immunoprecipitation and Western blotting of embryonic cells were performed following our previous protocol (35).…”